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. 1997 Jun 15;16(12):3544–3552. doi: 10.1093/emboj/16.12.3544

Modulation of murine melanocyte function in vitro by agouti signal protein.

C Sakai 1, M Ollmann 1, T Kobayashi 1, Z Abdel-Malek 1, J Muller 1, W D Vieira 1, G Imokawa 1, G S Barsh 1, V J Hearing 1
PMCID: PMC1169979  PMID: 9218796

Abstract

Molecular and biochemical mechanisms that switch melanocytes between the production of eumelanin or pheomelanin involve the opposing action of two intercellular signaling molecules, alpha-melanocyte-stimulating hormone (MSH) and agouti signal protein (ASP). In this study, we have characterized the physiological effects of ASP on eumelanogenic melanocytes in culture. Following exposure of black melan-a murine melanocytes to purified recombinant ASP in vitro, pigmentation was markedly inhibited and the production of eumelanosomes was decreased significantly. Melanosomes that were produced became pheomelanosome-like in structure, and chemical analysis showed that eumelanin production was significantly decreased. Melanocytes treated with ASP also exhibited time- and dose-dependent decreases in melanogenic gene expression, including those encoding tyrosinase and tyrosinase-related proteins 1 and 2. Conversely, melanocytes exposed to MSH exhibited an increase in tyrosinase gene expression and function. Simultaneous addition of ASP and MSH at approximately equimolar concentrations produced responses similar to those elicited by the hormone alone. These results demonstrate that eumelanogenic melanocytes can be induced in culture by ASP to exhibit features characteristic of pheomelanogenesis in vivo. Our data are consistent with the hypothesis that the effects of ASP on melanocytes are not mediated solely by inhibition of MSH binding to its receptor, and provide a cell culture model to identify novel factors whose presence is required for pheomelanogenesis.

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Selected References

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  1. Abdel-Malek Z., Swope V. B., Suzuki I., Akcali C., Harriger M. D., Boyce S. T., Urabe K., Hearing V. J. Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1789–1793. doi: 10.1073/pnas.92.5.1789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aroca P., Urabe K., Kobayashi T., Tsukamoto K., Hearing V. J. Melanin biosynthesis patterns following hormonal stimulation. J Biol Chem. 1993 Dec 5;268(34):25650–25655. [PubMed] [Google Scholar]
  3. Blanchard S. G., Harris C. O., Ittoop O. R., Nichols J. S., Parks D. J., Truesdale A. T., Wilkison W. O. Agouti antagonism of melanocortin binding and action in the B16F10 murine melanoma cell line. Biochemistry. 1995 Aug 22;34(33):10406–10411. doi: 10.1021/bi00033a012. [DOI] [PubMed] [Google Scholar]
  4. Hearing V. J., Jr Mammalian monophenol monooxygenase (tyrosinase): purification, properties, and reactions catalyzed. Methods Enzymol. 1987;142:154–165. doi: 10.1016/s0076-6879(87)42024-7. [DOI] [PubMed] [Google Scholar]
  5. Hunt G., Kyne S., Wakamatsu K., Ito S., Thody A. J. Nle4DPhe7 alpha-melanocyte-stimulating hormone increases the eumelanin:phaeomelanin ratio in cultured human melanocytes. J Invest Dermatol. 1995 Jan;104(1):83–85. doi: 10.1111/1523-1747.ep12613565. [DOI] [PubMed] [Google Scholar]
  6. Hustad C. M., Perry W. L., Siracusa L. D., Rasberry C., Cobb L., Cattanach B. M., Kovatch R., Copeland N. G., Jenkins N. A. Molecular genetic characterization of six recessive viable alleles of the mouse agouti locus. Genetics. 1995 May;140(1):255–265. doi: 10.1093/genetics/140.1.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ito S., Fujita K. Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical degradation and liquid chromatography. Anal Biochem. 1985 Feb 1;144(2):527–536. doi: 10.1016/0003-2697(85)90150-2. [DOI] [PubMed] [Google Scholar]
  8. Jiménez M., Kameyama K., Maloy W. L., Tomita Y., Hearing V. J. Mammalian tyrosinase: biosynthesis, processing, and modulation by melanocyte-stimulating hormone. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3830–3834. doi: 10.1073/pnas.85.11.3830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kobayashi T., Urabe K., Winder A., Jiménez-Cervantes C., Imokawa G., Brewington T., Solano F., García-Borrón J. C., Hearing V. J. Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis. EMBO J. 1994 Dec 15;13(24):5818–5825. doi: 10.1002/j.1460-2075.1994.tb06925.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kobayashi T., Vieira W. D., Potterf B., Sakai C., Imokawa G., Hearing V. J. Modulation of melanogenic protein expression during the switch from eu- to pheomelanogenesis. J Cell Sci. 1995 Jun;108(Pt 6):2301–2309. doi: 10.1242/jcs.108.6.2301. [DOI] [PubMed] [Google Scholar]
  11. Liggett S. B., Bouvier M., Hausdorff W. P., O'Dowd B., Caron M. G., Lefkowitz R. J. Altered patterns of agonist-stimulated cAMP accumulation in cells expressing mutant beta 2-adrenergic receptors lacking phosphorylation sites. Mol Pharmacol. 1989 Oct;36(4):641–646. [PubMed] [Google Scholar]
  12. Lu D., Willard D., Patel I. R., Kadwell S., Overton L., Kost T., Luther M., Chen W., Woychik R. P., Wilkison W. O. Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor. Nature. 1994 Oct 27;371(6500):799–802. doi: 10.1038/371799a0. [DOI] [PubMed] [Google Scholar]
  13. Manne J., Argeson A. C., Siracusa L. D. Mechanisms for the pleiotropic effects of the agouti gene. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4721–4724. doi: 10.1073/pnas.92.11.4721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McLane J., Osber M., Pawelek J. M. Phosphorylated isomers of L-dopa stimulate MSH binding capacity and responsiveness to MSH in cultured melanoma cells. Biochem Biophys Res Commun. 1987 Jun 15;145(2):719–725. doi: 10.1016/0006-291x(87)91024-2. [DOI] [PubMed] [Google Scholar]
  15. Michaud E. J., Bultman S. J., Klebig M. L., van Vugt M. J., Stubbs L. J., Russell L. B., Woychik R. P. A molecular model for the genetic and phenotypic characteristics of the mouse lethal yellow (Ay) mutation. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2562–2566. doi: 10.1073/pnas.91.7.2562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Millar S. E., Miller M. W., Stevens M. E., Barsh G. S. Expression and transgenic studies of the mouse agouti gene provide insight into the mechanisms by which mammalian coat color patterns are generated. Development. 1995 Oct;121(10):3223–3232. doi: 10.1242/dev.121.10.3223. [DOI] [PubMed] [Google Scholar]
  17. Miller M. W., Duhl D. M., Vrieling H., Cordes S. P., Ollmann M. M., Winkes B. M., Barsh G. S. Cloning of the mouse agouti gene predicts a secreted protein ubiquitously expressed in mice carrying the lethal yellow mutation. Genes Dev. 1993 Mar;7(3):454–467. doi: 10.1101/gad.7.3.454. [DOI] [PubMed] [Google Scholar]
  18. Mountjoy K. G., Robbins L. S., Mortrud M. T., Cone R. D. The cloning of a family of genes that encode the melanocortin receptors. Science. 1992 Aug 28;257(5074):1248–1251. doi: 10.1126/science.1325670. [DOI] [PubMed] [Google Scholar]
  19. Ozeki H., Ito S., Wakamatsu K., Hirobe T. Chemical characterization of hair melanins in various coat-color mutants of mice. J Invest Dermatol. 1995 Sep;105(3):361–366. doi: 10.1111/1523-1747.ep12320792. [DOI] [PubMed] [Google Scholar]
  20. Perry W. L., Copeland N. G., Jenkins N. A. The molecular basis for dominant yellow agouti coat color mutations. Bioessays. 1994 Oct;16(10):705–707. doi: 10.1002/bies.950161002. [DOI] [PubMed] [Google Scholar]
  21. Perry W. L., Nakamura T., Swing D. A., Secrest L., Eagleson B., Hustad C. M., Copeland N. G., Jenkins N. A. Coupled site-directed mutagenesis/transgenesis identifies important functional domains of the mouse agouti protein. Genetics. 1996 Sep;144(1):255–264. doi: 10.1093/genetics/144.1.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Prota G., Lamoreux M. L., Muller J., Kobayashi T., Napolitano A., Vincensi M. R., Sakai C., Hearing V. J. Comparative analysis of melanins and melanosomes produced by various coat color mutants. Pigment Cell Res. 1995 Jun;8(3):153–163. doi: 10.1111/j.1600-0749.1995.tb00657.x. [DOI] [PubMed] [Google Scholar]
  23. Sato C., Ito S., Takeuchi T. Establishment of a mouse melanocyte clone which synthesizes both eumelanin and pheomelanin. Cell Struct Funct. 1985 Dec;10(4):421–425. doi: 10.1247/csf.10.421. [DOI] [PubMed] [Google Scholar]
  24. Siegrist W., Willard D. H., Wilkison W. O., Eberle A. N. Agouti protein inhibits growth of B16 melanoma cells in vitro by acting through melanocortin receptors. Biochem Biophys Res Commun. 1996 Jan 5;218(1):171–175. doi: 10.1006/bbrc.1996.0030. [DOI] [PubMed] [Google Scholar]
  25. Siracusa L. D. The agouti gene: turned on to yellow. Trends Genet. 1994 Dec;10(12):423–428. doi: 10.1016/0168-9525(94)90112-0. [DOI] [PubMed] [Google Scholar]
  26. Suzuki I., Cone R. D., Im S., Nordlund J., Abdel-Malek Z. A. Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. Endocrinology. 1996 May;137(5):1627–1633. doi: 10.1210/endo.137.5.8612494. [DOI] [PubMed] [Google Scholar]
  27. Tsukamoto K., Jackson I. J., Urabe K., Montague P. M., Hearing V. J. A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase. EMBO J. 1992 Feb;11(2):519–526. doi: 10.1002/j.1460-2075.1992.tb05082.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vrieling H., Duhl D. M., Millar S. E., Miller K. A., Barsh G. S. Differences in dorsal and ventral pigmentation result from regional expression of the mouse agouti gene. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5667–5671. doi: 10.1073/pnas.91.12.5667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Willard D. H., Bodnar W., Harris C., Kiefer L., Nichols J. S., Blanchard S., Hoffman C., Moyer M., Burkhart W., Weiel J. Agouti structure and function: characterization of a potent alpha-melanocyte stimulating hormone receptor antagonist. Biochemistry. 1995 Sep 26;34(38):12341–12346. doi: 10.1021/bi00038a030. [DOI] [PubMed] [Google Scholar]
  30. Winder A., Kobayashi T., Tsukamoto K., Urabe K., Aroca P., Kameyama K., Hearing V. J. The tyrosinase gene family--interactions of melanogenic proteins to regulate melanogenesis. Cell Mol Biol Res. 1994;40(7-8):613–626. [PubMed] [Google Scholar]
  31. Yen T. T., Gill A. M., Frigeri L. G., Barsh G. S., Wolff G. L. Obesity, diabetes, and neoplasia in yellow A(vy)/- mice: ectopic expression of the agouti gene. FASEB J. 1994 May;8(8):479–488. doi: 10.1096/fasebj.8.8.8181666. [DOI] [PubMed] [Google Scholar]
  32. Zemel M. B., Kim J. H., Woychik R. P., Michaud E. J., Kadwell S. H., Patel I. R., Wilkison W. O. Agouti regulation of intracellular calcium: role in the insulin resistance of viable yellow mice. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4733–4737. doi: 10.1073/pnas.92.11.4733. [DOI] [PMC free article] [PubMed] [Google Scholar]

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